Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by 4 phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP4A reduced neurodegeneration caused by dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) ?mediated toxicity in dopaminergic neurons. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. On DIV8, cultures were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was further examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg), given subcutaneously every two hours. Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis.